The DNA cloning technology on which biotechnological development was based is a technique for amplifying circular DNA that had been prepared by cutting and pasting DNA fragments as plasmid in cells of E. coli, etc. A use of a DNA cloning technology that uses cells to amplify circular DNA necessitates troublesome procedures such as cell cultivation, extraction/purification of amplified products and the like. Also, the environment for experimenting such DNA cloning is limited, since it is necessary to prepare genetically modified organisms to perform DNA cloning that uses cells.
A common method used for amplifying DNA in vitro is polymerase chain reaction (PCR). However, an in vitro DNA amplification using PCR does not allow circular DNA to be amplified as it is. In vitro amplification methods of circular DNA include the rolling circle amplification (RCA) (Non-Patent Document 1, Patent Document 1, Patent Document 2, Patent Document 3). However, if circular DNA is to be amplified using the rolling circle amplification, a primer specific to the target DNA would need to be designed each time. Furthermore, the amplification product that directly results from the rolling circle amplification is a linear DNA, so it would be necessary to perform an additional cyclization step to cyclize the obtained amplification product, such as incubating with a recombinant enzyme. Another reported method is a method of obtaining a monomer replication product by separating a minichromosome of E. coli (oriC circular DNA) after it had been replicated (Non-Patent Document 2), but this method amplifies only circular DNA of up to about 6 kbp. There is also a report of rearranging replication of minichromosomes of E. coli using a group of purified enzymes derived from E. coli (Non-Patent Document 3), but no method is known for exponentially amplifying the entire long circular DNA in vitro by repeating replication cycles in a single test tube.
As shown above, amplification of circular DNA using the conventional in vitro DNA amplification was disadvantageous in that it required primers to be bonded with the template DNA, produced linear DNA as the amplification product, and limited the size of DNA that can be amplified to within a few kb.
Methods such as the SLIC method and the Gibson Assembly method have been proposed as technologies to prepare long DNAs, but in either case, the final preparation of the long chain circular DNA can only be performed by using cells (Non-Patent Document 4). A method using Bacillus subtilis has been reported as a method for preparing long chain circular DNA by using cells (Non-Patent Document 5).